项目名称: CRISPR/Cas9介导的染色体转位与剂量补偿效应研究
项目编号: No.81501329
项目类型: 青年科学基金项目
立项/批准年度: 2016
项目学科: 医药、卫生
项目作者: 冯雪竹
作者单位: 中国科学技术大学
项目金额: 18万元
中文摘要: 剂量补偿效应指的是在XY性别决定机制的生物中,使性连锁基因在两种性别中有相等或近乎相等的有效剂量的遗传效应,从而在雌性和雄性细胞里,由X染色体基因编码产生的蛋白质产物在数量上相等或近乎相等。X染色体上存在一些性别决定的序列元件,招募蛋白质因子和非编码RNA,来诱导异染色质形成和调节转录活性。剂量补偿效应的去调节往往会造成发育缺陷与疾病。我们希望在模式生物秀丽线虫里运用双sgRNA介导的CRISPR/Cas9技术诱导染色体易位,来研究染色体稳定性维持的机制以及性别代偿效应的机制。通过系统性地交换常染色体与性染色体片断,同时使用实时定量PCR检测常染色体与性染色体的基因表达水平,使用染色质免疫沉淀技术检测剂量补偿复合物在X染色体上的结合水平,来研究染色体微环境的改变对X染色体上基因表达的调控。这一研究有助于我们深入了解染色体微环境对基因表达的调控机制,也为人类染色体疾病研究带来新思路。
中文关键词: CRISPR;染色体;;易位;剂量代偿;性别决定
英文摘要: In many organisms sexual fate is determined by a chromosome-based method which entails a difference in sex chromosome-linked gene dosage. Consequently, a gene regulatory mechanism called dosage compensation equalizes X-linked gene expression between the sexes. There are many sequence elements localized on X-chromosome that recruit proteins factors and noncoding RNAs to direct heterochromatin formation and transcription regulation. Deregulation of dosage compensation results in developmental defects and human diseases. In this study, we propose to generate chromosome translocations by using dual sgRNA-directed CRISPR/Cas9 technology in C. elegans. We will use quantitative real time PCR to examine gene expression levels under different chromosome microenvironments, use chromatin immunoprecipitation assay to detect the association of Dosage Compensation Complex to X-chromosome. This study will help us to understand the mechanism of gene regulation by chromosomal microenvironments, and facilitate our study of human diseases.
英文关键词: CRISPR;chromosome;translocation;dosage compensation;sex determination